Method for the production of microbial insecticides



p 23, 1963 B. J. MECHALAS 3,086,922

METHOD FOR THE PRODUCTION OF MICROBIAL INSECTICIDES Filed Oct. 15, 1961WATER STEAM I u A 5 a e. '5 E. A 1

2. FILTER HUMIDIFIER L STEAM HEAT 17 '8 EXCHANGER BIN FOR 0 SEMI SOLID25 HUMIDITY CONTROLLER AIR J HEAT VOLUME 35 2' EXCHANGER REL-ORDERCHILLED WATER TEMPERATURE CONTROLLER E INVEN TOR.

A TTORNE YS United States Patent 3,tl86,922 METHOD FOR THE PRODUCTION 0FMICRGBIAL INSECTICEDES Byron J. Mechalas, San Jacinto, Calif, assignorto Nutrilite Products, Inc., Buena Park, Calif., a corporation ofCalifornia Filed Oct. 13, 1961, Set. No. rasnaz 18 Claims. (Cl. l9596)This invention relates to microbial insecticides. More particularly, theinvention relates to a novel microbial insecticide and to the productionand utilization thereof.

This application is a continuation-in-part of co-pending applicationSerial No. 4,543, filed January 25, 1960, and now abandoned.

The effective control of insects presents a constant and perplexingproblem. The present annual expenditure in the United States forchemical insecticides alone is approximately one hundred milliondollars. Chemical insecticides are inherently undesirable in manyrespects. The high order of toxicity toward forms of life other thaninsects which characterizes most chemical insecticides is seriouslyobjectionable. The toxicity problem presents a hazard, not only toworkers who must deal directly with the toxic insecticidal compositions,but also to the ultimate consumer of treated agricultural products whichmay be expected to carry residual quantities of the insecticide. Thehazard of residual insecticide toxicity is particularly acute for wantof an effective method of control by Government authorities. Mostchemical insecticides are also disadvantageously non-specific, and hencekill not only undesirable insect pests, but also large numbers ofbeneficial insects such as pollinators and predators. The normalentomological balance of nature is accordingly disturbed. Moreover,insects frequently develop resistance to chemical insecticides,necessitating periodic formulation and type changes.

Microbial control of insects offers significant advantage in comparisonwith chemical insecticides. Insect pathogens are harmless and non-toxicfor other forms of life, and hence leave no objectionable residues.Microbial insecticides demonstrate a relatively high degree ofspecificity and hence tend to protect beneficial insects, such aspollinators, parasites, and predators. Moreover, a susceptible insecthost develops resistance to microbial pathogens only quite slowly, if atall. Microbial insecticides are effectively applied as dusts or sprays,may be introduced or colonized, may be used with chemical insecticides,both compatibly and synerg-istically, and may be used in combinationwith parasites and predators. Low dosages are required.

Diseases of insects, now known to be caused by microorganisms, includingthose which attack the honeybee and the silkworm, were familiar beforethe time of Aristotle. Early efiorts to protect such beneficial insectsyielded some comprehension of the nature of insect infectious diseases.Bassi in 1834 proved that a fungus disease of the silkworm could becaused by a microorgamsm.

Utilization of infectious diseases to control insect pests has longintrigued the art. In 1879 Metchnikoif succeeded in infecting larvae ofthe wheat cockchafer by inoculating a pot of soil containing insectswith a pathogenic fungus. The success attained by Metchnikoflf inspiredKrasilshchik of the University of Odessa to establish a laboratory in1884 for the purpose of producing spores of the same fungus on a largescale.

Berliner, in 1911, published a report in Germany con.- cerning a diseaseof the larvae of the Mediterranean flour moth caused by a spore-formingbacterium, later named Bacillus thuringiensis.

3,086,922 Patented Apr. 23, 1963 ice In this country, interest in themicrobial control of insects stimulated the establishment under Dr.Edward A. Steinhaus of a laboratory at the University of California in1945, to investigate the possibility of the microbial control of thealfalfa caterpillar. In 1956, Dr. Steinhaus recognized that much of whathad been accomplished in the way of microbial control of insects hadbeen based on trial-and-error methods, and that adequate fundamentalknowledge on which to base microbial control procedures had not beendeveloped. Dr. Steinhaus, in 1956, was of the view that the developmentof a satisfactory method for the large scale commercial production ofsuch microbial insecticides was a problem of primary significance.

It is a primary object of this invention to provide a novel and highlyefficacious method for the production and, more particularly, thecommercial production of a microbial insecticide.

It is an additional important object of the invention to provide asurface culture technique for the production of a microbial insecticide.This technique is designed to retain all toxic components both solubleand insoluble.

It is an additional primary object of the invention to provide amicrobial insecticide comprising the entire culture including thespores, soluble and insoluble metabolic by-products of the Bacillus andthe residue of the nutrient medium on which Bacillus thuringiensis isgrown and sporulated.

The preservation of all of these ingredients provides for a microbialinsecticide of a potency substantially higher than that produced whenthese products are discarded during processing by more conventionalmethods.

It is also an object of the invention to provide a method for themicrobiological control of insects which contemplates utilization as amicrobial insecticide of the entire culture including all metabolicby-products and including the residual nutrient medium on which Bacillusthuringiensis is grown and sporulated.

It is a more specific object of the invention to provide a method usefulfor the production on a, commercial scale of a microbial insecticidecontaining spores of Bacillus thwringiensis.

It is an additional object of the invention to provide a methodeflfective to provide a microbial insecticide derived from Bacillusthuringiensis characterized by an organism and spore count higher thanachieved by certain prior art methods for producing such insecticides.Included in this will be the entire bacterial culture, its metabolicproducts and the residual nutrient medium. This results in a product ofunusually high potency; soluble components are not lost duringprocessing.

A further object of the invention is to provide an improved method forthe microbiological control of insects including larval stages ofLepidoptera and Diptera.

A more specific object of the invention is to provide a method feasiblefor the production of microbial insecticides derived from Bacillusthuringiensis in a quantity adequate for the commercial control ofLepidoptera and Diptera.

I have discovered that the composition produced by my method ischaracterized by the presence of a heat stable, soluble component toxicto insects and more specifically toxic for larvae of diptera. It is theinclusion of this component that contributes to the unusually highpotency of the products of this invention.

It is another object of the invention to provide a novel apparatususeful for the commercial large-scale production of a microbialinsecticide by the propagation and sporulatiou of Bacillusthuringiensis.

The biological insect control method of the invention contemplatesfeeding the insects to be controlled the entire culture of sporulatedBacillus thuringiensis, as distinguished from an extract or sporeconcentrate derived acsasaa from such a culture as in the prior art.Accordingly, the invention provides a semi-solid, surface culturepropagation technique effective to provide product comprising the entiresporulated culture, which is of requisite toxicity to be useful in thecommercial control of insects.

More specifically, the invention provides a microbial insecticide ofhigh spore content and potency produced by inoculating the surface of anutrient medium with Bacillus thuriugicnsis, passing first stage airinto said inoculated medium at a pressure of not more than about fivepounds per square inch and at a rate of from about 0.2 to about 1.2, andpreferably about 1.0 volume of air per volume of medium per hour untilsubstantial sporulation occurs, said first stage air being introducedinto said medium at :a temperature of from about 25 C. to about 35 C.and at a relative humidity of at least 95%, maintaining said medium at atemperature of from about 25 C. to about 35 C. substantially throughoutthe period said first stage air is passed therethrough, and thereafterpassing second stage air having a relative humidity substantially lowerand a temperature not in excess of 60 C. but substantially higher thansaid first stage air through said sporulated nutrient medium to reducethe moisture content thereof to not more than about by weight. Duringthis latter step most of the moisture is taken off by evaporation withonly a negligible loss of the water soluble products of thefermentation.

The art is familiar with nutrient media useful for the propagation ofBacillus thuringiensis. Such media appro- .priately contain protein andcarbohydrate materials and may conform to those disclosed in Prescottand Dunn, Industrial Microbiology, McGraw-Hill, 195, pp. 68, 73, 90,50l507, and 527. Alternatively media consisting essentially of essentialmineral-, vitamin-, and nitrogen-supplying materials may be utilized,such as are present in ordinary nutrient agar, as disclosed bySteinhaus, Hilgardia 20, p, 368 'et seq. (195 1). A nutrient mediumfound to be particularly useful in the production of the microbialinsecticides contemplated by the invention comprises:

Weight, percent Soy bean meal 30 to 40 Sugar (eg. dextrose) to Fish meal15 to 20 Dried milk 15 to 20 Bacillus thuriugiensis, in common withBacillus species generally, is an amylase producer. Accordingly, in lieuof carbohydrates other than starches, an appropriate nutrient medium maycomprise a mixture of bran and expanded perlite. Bacillus thuringiensfs,in utilization of such a medium produces amylase which, in turn, breaksdown the starch content of the bran to assimilable carbohydrates such assugars. In a preferred embodiment, the nutrient medium is adsorbed ontoa particulate inorganic carrier and nutrient substrate. Appropriateorganic substrates include bran, wheat middlings, red dog flour, alkalfameal, corn meal, peanut meal, oat hulls, rice hulls oatmeal, cornstalks, corn cobs, kudzu vines, sorghum stalks, beet pulp, soybeanvines, sweet potato vines, sweet potatoes, Irish potatoes, cottonseedmeal and the like. Vegetable materials utilized as a carrier for or as apart of the nutrient medium are preferably comminuted to provide a highratio of surface area to volume and hence encouage vigorous bacterialgrowth. Experimental evidence indicates that organic carrier materialssuch as bran afford appreciable quantities of nutrients to Bacillusthuringiensis.

Utilization of inorganic carriers affords a microbial insecticideculture which may be comminuted to a selected mesh size more readilythan comparable cultures propagated on media composed entirely fromorganic materials. Preferred inorganic carriers include expandedvolcanic glasses such as perlite, obsidian, and the like; exfoliatedvermiculite, pumice, volcanic ash, calcined diatomaceous earth, andsimilar materials preferably characterized by i a substantial degree offriability requisite to facilitate comminution of microbial insecticideculture.

Optimum results are obtained with mixtures of inorganic and organiccarrier materials. The invention generically contemplates such mixturesin all relative proportions. Preferred mixed carrier media comprise fromabout 20% to about by volume of inorganic material and about 80% toabout 20% by volume of organic material. A particularly appropriatecarrier medium coniprises from about 20% to about 80% by volume ofexpanded perlite and about 80% to about 20% by volume of an organicmaterial such as bran.

Microbial insecticides are appropriately produced in accordance with themethod of the invention in an ap' paratus such as that schematicallyrepresented in FIG URE 1. Referring to the drawing, air is passedthrough line r10, filter 11 and line 12 into humidifier :13 which isprovided with means 14 for the controlled introduction of steam andmeans '15 for the controlled introduction of water. Humidified airpasses from humidifier 113 through line 16 into heat exchanger 17 whichis provided with means 18 for the controlled introduction of steam.Heated and humidified air passes from heat exchanger 17 through line 19into blower 20 and thence through line 21 into heat exchanger 22 whichis provided with means 23 for the controlled introduction of chilledwater. From heat exchanger 2'2, the air passes through line 24 intohumidity controller Q5. Properly heated and humidified air exits fromhumidity controller 25 through line 26, air volume recorder 27, line 28,control valve 29', and line 30 in bin 31.

Bin 31 is constructed and arranged for the effective growth andsporulation of Bacillus thuringiensis, and includes a lower air chamber32 separated by foraminous partition 33 from an upper propagationchamber 34 which contains inoculated nutrient medium when the apparatusis in use. The arrangement is such that air, at the proper temperatureand humidity, is passed upwardly from chamber 32 through foraminouspartition 33 into and through inoculated nutrient medium contained inpropagation chamber 34.

Temperature controller '35 is operatively connected by lines 36, 37, =38and 39, respectively, to temperature probes, not shown, positioned incontrol means 18, 23, and 29 and to temperature probe 40' positioned inchamber '34. Humidity controller 25 is operatively connected by line 41to control means 14.

In general, [the preparation of a microbial insecticide as contemplatedby the invention entails the steps of (a) reparing an inoculant of theBacillus thuringiensis microorganism to be utilized; (b) inoculation ofa nutrient medium with the prepared inoculant; (c) propagation of theinoculated medium; (d) reducing the moisture content of the propagatedmedium to a proper low level; (e) comminuting the dried propagatedmedium and metabolic products to a particle size appropriate forutilization as an insecticide.

In the preferred practice of the invention, the inoculant is prepared intwo steps, i.e. a small flask culture is first produced and utilized inthe preparation of a comparable culture in significantly larger volume.

The preparation of a flask culture entails the initial steps of placingthe appropriate nutrient medium in a flask, and inoculation of suchmedium with a pure Bacillus thurz'ngiensis culture, such as may be grownin conventional manner in a slant tube. An appropriate flask culturemedium may contain the following ingredients:

Percent by weight Water to 98 Corn steep 0.4 to 2.0 Dextrose 1.0 to 3.0Yeast autolysate 0.25 to 1.0

Sodium hydroxide to pH 7.

, The nutrient medium is heat sterilized, preferably for at leastminutes, cooled to to C., inoculated with pure Bacillus thuringiensisculture from a slant tube and placed on a shaker for about 8 hours at atemperature of about 28 C. to 30 C. If satisfactory growth is evidencedat the end of the 8-hour period, the flask culture may be transferred toan inoculating bottle.

The seed-tank culture is prepared normally in a large tank. A nutrientmedium of the same type as that utilized with the flask culture ispreferably employed. Corn oil or any other suitable anti-foam agent maybe introduced, if desired, to control foam. The medium in the tank issterilized for 20 minutes at 120 C., cooled and inoculated with theflask culture and agitated with air at 25 C. to C., e.g. at 30 C. forabout 20 hours. The seed-tank culture is utilized to inoculate aparticulate nutrient substrate of the type previously described.

As a specific example, a substrate material, such as bran or expandedperlite, is coated or impregnated with additional nutrient materials inapproximately the following proportions, by weight:

Percent by weight Bran or perlite 85 to 90 Soy bean meal 4.5 to 7 Sugar(dextrose) 3 to 5 Lime 0.3 to 0.6 CaCl;, .02 to .04 NaCl .07 to .11

Water is added to the foregoing mixture to a moisture content of about15% to 20% by weight, and to provide a pH of from about 6 to about 8,the pH being adjusted to about this range, if necessary. The mixture issteam sterilized and cooled to a temperature of about 37 C.

Seed-tank inoculant is then sprayed onto the nutrient carrier substancepreferably until the moisture content of the mixture is within the rangeof from about 50% to about 6 5% by weight. The inoculated nutrientmaterial is transferred to a bin of the type described in reference toFIGURE 1. First stage air at a temperature of about 28 C. to about 34 C.is introduced at a relative humidity of at least 70% and preferablyabout 80% to about 100%. The introduction of first stage air under theaforementioned conditions of temperature and humidity is continued untilsubstantial sporulation has occurred. Normally, such sporulation isachieved within a .time period from about 30to about 48 hours. Secondstage air is then introduced at a temperature of about 45 C. to about 60C. and at a low relative humidity for a time period requisite to reducethe moisture content of the sporulated material to not more than about10%, and preferably to from about 3% to about 6% by weight. Normally, aproduct is satisfactorily dried after second stage air has beenintroduced for about 40 hours.

If desired, the sporulated nutrient medium and carrier substrate may beagitated to break down the material and expedite drying, prior to orduring contact with second stage air.

The dried sporulated product may be comminuted to a particle sizeappropriate for utilization as a dust or a wetta'ble powder, inadmixture with chemical insecticides, or the like. In general, it ispreferable to effect comminution at a temperature not in excess of about60 C. to a particle size of -80 mesh. Comminution at temperaturessubstantially in excess of 60 C. may materially reduce the spore countof the microbial insecticide prodnet.

The particle size is not critical. However, it should be such as toprovide the most eflicient distribution or coverage of the areastreated. At the same time it should not be so fine as to bring aboutmechanical problems especially when applied as a dust. The size willdepend largely upon how the material is to be used. Obviously, if it isto be mixed With a liquid and sprayed on, a finer 6 powder may beadvantageous than where it is applied as a dust.

To summarize, the inclubation period should be at least about 30 hoursand preferably about 35 hours at temperatures of about 25 C. to 35 C.and preferably about 28 C. to about 34 C. For periods above about 35hours of incubation, little change in spore count occurs.

The relative humidity is preferably about 95% or more, 97--.100% beingoptimum, for the first stage air employed during the incubation period.The function of the moist air is to maintain the moisture content of thesubstrate at the optimum moisture content of about 55-62% of theinoculated substrate and also to aid in active sporulation. At lowrelative humidities the rate of sporulation is adversely affected eventhough the moisture content of the particles is above 50%.

The substrate may be inoculated with tank inoculum or with dried spores.When the dried spores are used, it is added just before the mass isplaced in the sporulation bins and the moisture is then adjusted.Preferably a temperature of about 50 C. is used for inoculation with thedried spores since this appears to heat shock the spores, initiating amore rapid germination. Approximately one pound of 150 BSG material isused per 300 pounds of substrate on a dry basis. .After inoculation, thetemperature is dropped to approximately about 28 to 34 C. With eitherWet or dry inoculation it is advantageous to carry out the earlyportions of the sporulation at a temperature of about I34 or 35 C. andlater the temperature may be dropped to about 32 C.

The air employed for the second stage should have a substantially lowerrelative humidity than that used in the first stage. It is preferably ofthe order of -10 to 20% and advantageously should be well below 50 to60%. The temperature of the second stage treatment should be above 35and preferably of the order of 50 to 55 C. This promotes more rapiddrying. The process is best carried out both in the first and secondstages as rapidly as possible so as to eliminate or minimize thepossibility of contamination of the material with other organisms. Careshould be taken to use clean air with as little contamination aspossible with other organisms which might develop in the massparticularly during the sporulation and early portion of the dryingstages.

EXAMPLE I A. Preparation of Inoculunt Culture Approximately 100 ml. of anutrient medium of the 7 with sodium hydroxide, heat-sterilized andcooled to 28 C. The sterilized medium was then inoculated with a pureculture of Bacillus thuringiensis from a slant tube,

' and the flask was placed on a shaker for eight hours to produce aflask culture from which a seed-tank culture was produced.

A seed tank was filled with eighty gallons of a nutrient medium having apH of 7.1 and consisting of 16 pounds of dextrose, eight pounds of cornsteep, four pounds of peptone, twelve ounces of sodium hydroxide and twoliters of corn oil as a foam-control agent. The medium in the seed tankwas sterilized for twenty minutes at 120 C. and 15 p.s.i. pressurecooled to about 30 C. and inoculated with ml. of the flask culture ofBacillus thuringiensis. T he pH of the inoculated seed-tank culture was6.7. Air,

at a pressure of about 3 pounds per square inch, at a temperature ofabout 30 C. was introduced into the inoculated seed-tank culture forabout 16 hours at a rate of about 3 volumes per volume of culture perhour. Ac-

average temperature of the inoculated medium during the passage of thefirst stage air therethrough was 343 C. The moisture content of thesporulated medium at the termination of the introduction of first stageair was about tive growth of the Bacillus thuringiensis was evident. The60% by weight. pH of the seed-tank culture after #16 hours was 5.1. TheThereafter, second stage air having substantially the eighty gallons ofseed-tank culture so produced provided same properties as the secondstage air employed in Exthe inoculant culture for the production of themicrobial ample I was passed through the sporulated medium forinsecticide. about 41 hours to provide a rfinal product having a mois B.Preparation of Microbial Insecticide ture content of about 5% and aspore count of 659x10 Two hundred eighty-three and three-fourths poundsof spores per gram a nutrient medium having a pH of 6.6 and consistingof The commmlltd to 95% through 80 mesh the 011 0 wing ingredients wasPrepared: Aqueous dispersions contaming 1, 0.5 and 0.25 gr. of

the comminuted material per 100 m1. of water were pre- Wheat bran 225pared. Alfalfa leaves were dipped into each of the aque- Soy bean mealous dispersions and fed to larvae of the alfalfa caterpillar, Dextrose 5Coleus philodice eurytlzeme. Tests were replicated five plsh merill i'itimes using 5 larvae per group to provide a total of Dned mllk,twenty-five larvae per sample. The results are reported Hydrated lime inTablel Tap water "ga o 5 TABLE 1 The nutrient medium was heat sterilizedfor sixty minutes, cooled to about 30 C., and inoculated with 40 gal-Percent mortality lons of the seed-tank culture. The pH of theinoculated pore ount Concentration medium was 6.35, the moisture contentwas 5 7% by 'Day 1 Day2 Day3 Day 4 weight, and the ambient relativehumidity was 38%. About one hour subsequent to inoculation, the temper;-659x10 1 0 2 50 94 100 ture of the medium had risen to 37 C. indicatinggrow 015 gb l IHL 0 0 62 98 of Bacillus thuringiensis had started. 100 00 68 96 First stage air was then introduced into the medium at h arelative humidity of about 70% to about 100% at an EXAMPLE HI averagetemperature of about 25 C. to about 30 C. A microbial insecticideproduct produced in a manner and at an average rate of from about 0.2 toabout 0.3 comparable tov that described in Example II but in a totalvolume per volume of inoculated medium per minute. processing time ofabout 50 hours, first stage air being The introduction of first stageair was continued for introduced for twenty-one hours and second stageair for 47 hours at which time the pH of the inoculated mediumthirty-nine, demonstrated a spore count of 4.17 X 10 per was 7.0,moisture content was 44% by Weight, and subgram. stantial growth andsporulation of the Bacillus thurin- Insect mortality tests wereconducted in the same mangiensis was apparent. Throughout the 47 hourperiod ner and with the same type of insect as described in during whichfirst stage air was introduced, the tempera- Example I I. The resultsare reported in Table 2. ture of the inoculated medium was maintained atan TABLE2 average of from about 28 C. to about 30 C.

Introduction of second stage air was dry as possible Percent mortalityand having an average temperature of from about 40 C. Spore countConcentration to about 55 C. was then initiated and continued for 45Dayl Dayz Days Dan about 46 hours to produce a sporulated product havinga moisture content of 4% by weight, and a pH of 7. The x 1 0 0 78 98product which demonstrated spore count of 9.15 10 8 0 g3 g3 spores pergram and was comminuted to 95 minus 80 50 mesh to provide an excellentmicrobial insecticide. It is apparent from a comparison of the data ofTable 1 EXAMPLE H with the data of Table 2 that the toxicity of themicrobial Following the Same Prooedllre and lltililing 252 P0111168insecticide products of the invention may vary directly of startingmaterials of the same composition in subwith h spore count, stantiallythe same relative proportion as described in Example I, 171 pounds ofmicrobial insecticide was pro- EXAMPLE duced. First stage air wasintroduced for about 27 hours A series of microbial insecticide productswere preunder the same conditions of temperature and humidity pared byprocedure described in reference to Example I, and at the same rate asdescribed in Example I. The with specific conditions and results asindicatedin Table- 3. TABLE 3 Age of Amt. of Age of Amt. Semi-Semiln0c., 11100., V01. seed pH seed used, Medium Amt. pH pH after solidsolid hrs. m1. tank, gal. tank, gal. start start moist., temp hrs.percent C BT-901 24 100 7.3 19% 40 Std 284 9.0 7.0 59 a2 9.4 6.7 asBT-902 '72 10 7.3 19% 40 Std 284 9.2 7.0 59 30 9.5 6. 7 36% BT-903 48250 80 7.2 20 40 Std--.-- 284 10.2 6.5 59 31 as as BT-905 72 100 so 7.219% 40 Std 284 10.0 5. 58 BT-906 72 100 80 7.2 19% 40 Std--." 284 10.159

TABLE 3-Continued Time to Rel. Rel. Bran Time of Change start Time ofTime to Change humidhumidmoisture, Temp, Temp. Spore of pH growth,growth dry pH Yield ity, 1-5 lty, 40-48 inc. to inc., count hrs. hr.,percent hrs., 0 hrs. X10 9 percent percent Bio-assays of the microbialinsecticide products of this example were conducted as follows:

Insectary reared larvae of the salt marsh caterpillar, Estigmcna acrea(Dru.), were employed as test insects. The host insects were eight daysold, the day of batch being regarded as the first day, when subjected tothe test. The host insects were selected for uniform size and healthyappearance.

Fifty larvae were used for each microbial insecticide product tested.For each test, ten one-half pint cardboard food containers covered witha standard 110 mm. petri dish bottom and containing five larvae wereutilized.

Each microbial insecticide product was tested in concentrations of 0.1gram and 0.2 gram per 100 ml. of water prepared by blending of theproduct with water in a Waring Bl-endor, followed by separation andappropriate dilution with water.

The test insects were fed fibouquets of alfalfa made by securing with acotton plug the stem end of a single three-inch long sprig of alfalfa ina 50 mm. x mm. vial of water. The bouquets of alfalfa were dipped intobeakers of the material being tested and agitated until thoroughly wet.The bouquets were then air dried and one bouquet added to each carton offive larvae.

The larvae were allowed to feed on the treated alfalfa for four days;after the feeding period the number of dead and live larvae in eachcontainer was recorded. If a larva did not respond to tactilestimulation, it was regarded as dead. It a larva was moribund butexhibited some movement when prodded, it was regarded as alive. Seventypercent mortality was regarded as indicative of an acceptable microbialinsecticide product. The test results are reported in Table 4.

TABLE 4.-'IOXIOITY Spore Spores Percent Spores Percent Sample count 100ml. mortality, 100 ml. mortality,

l0/gm x10 1 10 gm x10 1 5 gm./

100 ml 100 m1.

EXAMPLE v This example is included for comparative purposes to reflectthe nature of the product obtained when aeration techniques whichcharacterize the process of the invention are not employed.

One hundred twenty-one pounds of a medium consist ing of 100 pounds ofbran, 10 pounds of soy bean meal, 10 pounds of sugar and one pound ofsodium hydroxide was sterilized with steam for ninety minutes and cooledwith air to about room temperature. sterilized medium Was 10.8.

The sterilized medium was inoculated with twenty gallons of a seed tankculture Bacillus thuringiensis comparable to that described in ExampleI. The inoculated medium was spread in four trays and maintained at atemperature of about 30 C. to facilitate propagation and sporulation ofthe Bacillus thuringiensis. Three of the trays were dried for 18 hoursand the fourth tray was dried for 24 hours.

No effective sporulation was achieved. On a bio-assay of the typedescribed in Example 11,. the products of this example, in aconcentration of twenty-five grams per 100 milliliters of waterdemonstrated only 4% mortality after four days. In a concentration of12.5 grams per 100 milliliters of Water, no mortality was demonstrated.

It will be appreciated that the expression Bacillus thuringiensis isemployed generically in this specification to embrace all species andsubspecies of thuringiensis, including specifically but withoutlimitation, Bacillus thurin- In general, recently developed proceduresfor the production ofmicrobial duction of microbial insecticides fromBacillus thuringiensis entail submerged culture techniques which requirecostly equipment and process expedients. In contrast the surface culturetechnique of this invention aifords an effective microbial insecticideproduct at comparatively low cost.

The pH of the EXAMPLE VI This example is illustrative of the productionof a microbial insecticide through utilization of a culture mediumcomprising a mixture of organic and inorganic carrier materials andconforming the following formulation:

pared in the manner described in Example I. The inoculated culture wasthen processed in a manner corresponding to that described in Example I.More specifically the median temperature was held at about 30 C.throughout the major portion of the growth phase; a temperature range ofabout 30 C. being maintained for of the growth period. The relativehumidity of the air stream utilized was maintained at about Moisturecontent of the culture was initially 56% by weight, 51% by weight after24 hours and 46% by weight after 48 hours,

at which time the growth phase was terminated and the culture dried asdescribed in Example I. The dried material was ground throughsuccessively finer screens in a Fitz-mill to produce a much finer meshmicrobial insecticide product than can be produced from a bran mediumalone under like conditions. Five fractions were separated as follows:

(1) 40 mesh All material passed. (2) 80 mesh 70% passed. (3) 100 mesh50% passed. (4) 150 mesh 23% passed. (5) 200 mesh passed.

The spore count of the entire ground material was 12.6 10 spores pergram. The spore count of the several fractions of material retained oneach screen Was as follows Spores/gram #1. 80 mesh (retained) 8.5 1O #2.100 10.1)(10 #3. 150 9.2)(10 #4. 200 9.7)(10 #5. Passed 200 9.3)(10Standard bio-assays against the salt marsh caterpillar was carried outin the manner generally described in Example 4, demonstrated theeffectiveness of the microbial insecticide product as reflected by thedata reported in Table 5.

TABLE 5 a. 0.2 gm./100 ml. water 1 0.1 gu1./l00 ml. Water 0 0.05 gm./l00ml. water Dosage Percent mortality ET 927, entire material BT 927-1, 80(retained) BT 927-2, 100 (retained) BT 927-3, 150 (retained) BT 927-4,200 (retained) BT 927-5, 200 (passed) Standard 9001-A Control 0.01gm./100 ml Untreated The foregoing example demonstrates that theutilization of a mixed organic and inorganic carrier medium resulted ina microbial insecticide product of excellent spore count. Such inorganiccarrier containing media normally yield a product containing from about30% to about 50% higherspore count than products produced on organicmedia such as bran, in the absence of inorganic carrier materials.Moreover, the inorganic component of the medium precludes packing of theorganic component and hence ailords improved air flow. Inorganicmaterials such as perlite which are friable additionally appear to actas grinding aids and facilitate production of microbial insecticideproducts of high toxicity at various mesh sizes.

EXAMPLE VII This will demonstrate the increased potency both as to sporecount and insect toxicity obtained when all of 12 the conditions andimprovements described in the preceding material is critically adheredto during processing.

A. Preparation 07 Inocalant Culture Approximately 100 ml. of a nutrientmedium of the following composition was placed in each of 4-500 ml.flask.

Percent by weight Water 97.1 Corn steep 0.5 Sugar 1.5 Yea-st autolysate0.5 K HPO 0.4

The nutritive medium in the flasks was adjusted to pH 7.2 with sodiumhydroxide, heat-sterilized, and cooled to 30 C. The sterilized mediumwas then inoculated with a pure culture of Bacillus thuringiensis sporesfrom a soil stock culture, and the flask was placed on a shaker foreight hours to produce a flask culture from which a seed tank culturewas produced.

A seed tank was filled with one hundred gallons of a nutrient mediumhaving a pH of 7.2 and consisting of nine pounds of dextrose, fourpounds of corn steep liquor, five pounds of yeast autolysate, six ouncesof sodium hydroxide, three pounds of di-potassium phosphate, andthirty-eight grams of calcium chloride. Foam was controlled through thecontinuous addition of a commercial antifoam compound. The medium in theseed tank was sterilized for twenty minutes at 120 C. and 15 p.s.i.pressure, cooled to about 30 C. and inoculated with 400 ml. of the flaskculture of Bacillus tharingiensis. The pH of the inoculated seed tankwas 6.9. Air at a pressure of about 5 psi. at a temperature of about 30C. was introduced into the inocuated seed tank culture for about sixteenhours at a rate equivalent to a sulfite oxidation value of 1.5(millimoles of oxygen absorbed per milliliter of medium per minute).Active growth ofBacillas thuringiensis was evident. The pH of the seedtank culture after sixteen hours was 7.0. The one hundred gallons ofseed tank culture so produced provided the inoculant culture for theproduction of a microbial insecticide.

B. Preparation of Microbial Insecticide Two thousand two hundred andsixty-seven pounds of a nutrient medium having a pH of 6.8 andconsisting of the following ingredients was prepared:

Wheat bran pounds 1200 Expanded perlite do 840 Soybean meal do 136Dextrose do Lime do 8 Sodium chloride do 2 Calcium chloride "grams" 28840 The nutrient medium was heat sterilized for sixty minutes, cooled to32 C. and inoculated with 100 gallons of the seed tank. The pH of theinoculated medium was 6.9, the moisture content 60% by weight, and theambient relative humidity was 36%. This was divided equally into fourbins.

First stage air was then introduced into the medium at a relativehumidity of about to about at an average temperature of about 30 C. toabout 34 C.,

and at an average rate of about 0.4-0.6 volume per volume of inoculatedmedium per minute; this was increased to 1.0-1.2 volumes after aboutthree hours. The introduction of first stage air was continued for about36 hours at which time the pH of the inoculated medium 13 was 7.5, themoisture content was 53% by weight and substantial growth andsporulation of Bacillus thuringiensis had occurred; Throughout thethirty-six hour period during which first stage air was introduced, thetemperature of the culture was maintained at an average of from about 31C. to about 33 C.

The bins were then shifted to the drying side. Second stage air as dryas possible and having an average temperature of from about 50 C. toabout 55 C. was then initiated and continued for about thirty-six hoursproduced a sporulated product having a moisture content of about 4% byweight and a pH of 7.0. The prodnot which demonstrated a viable sporecount of 234x10 spores per gram and was comminuted to 95% minus 80 meshto provide an excellent microbial insecticide.

EXAMPLE VIII This example is presented to illustrate the presence of ahitherto unsuspected heat stable soluble component that is toxic wheningested by certain insects, especially Diptera. Furthermore, thissecond component is wholly retained in the process of the invention asopposed to more conventional fermentation procedures where thiscomponent is discarded in the supernatants and wash liquors. Theretention of all metabolic products as developed by the process of thisinvention yields an insecticide of superior potency on a spore basis andalso extends the host range to cover a wider spectrum of insects. Thefollowing data illustrate these points:

ASSAY ORGANISM-HOUSE FLY [Sample B'I 9019-Iroduced in conventionalfermentation equipment 1 Percent Dosage spores/gram mortality Solidsfrom fermenter: Washed and dried (baterial spores-no soluble residue)Washed and autoclaved Turbofilm processed (spores and some solubleresidue).

0.19 10 2.0 m1./100 gm. medium.. 1.0 IIlL/IUO gm. medium-.- 0.5 m1./100gm. medium.-- 025 m1./100 gm. medium-. 2.0 nil/100 gm. medium.. 1.0ml./100 gm. medium... 0.5 ml./100 gm. medium... 0.25 ml./l gm. mediu.m

Supernate from termenter, as

Autoolaved cocnqucecnwq r-n-noe-u-n-noeuimeooamcsoaoowiaowoowoomcooo[Sample B'I 5005-Produeed by means of this invention] As is 1.5Xl00.75X. 0.37 X10 010x10 Untrea ted Autoelaved Control- ASSAYORGANISM-SALT MARSH CATERPILLAR [Sample BT 9019-Produced in conventionalfermentation equipment] [Sample BT SOUS-Prepared by process of thisinvention] AS iS 1.5)(10 92 52 Autoclav 1.5X10 7 0 75x10 2 (hankUntreated 2 Here the presence in the solids as well as in the supernateof a heat labile factor toxic to the caterpillar but not to the housefly is shown. The toxic factor of conventionally produced insecticidewhich is present in the solids was heretofore discarded. But there ismore to the present invention-than the mere retention of all componentsas may be seen from the data. Not only is a product containing bothtoxic components produced, but the potency of the material is increasedover that of the material produced by the conventional method.

In the autoclaving tests the material was held for ten minutes in anautoclave heated by steam at 15 p.s.i.g. Unless otherwise noted, thepercent mortality was determined after four days.

In preparing the substrate or carrier, the material may be of largermesh size than that for the final product. While it should not be toocoarse, the larger particles and absence or minimization of finesrenders the bed more open and hence provides for greater uniformity ofair distribution. For example -8 +20 mesh is a suitable mesh size. Ifthe air pressure is not too high, channeling is avoided. Wheresubstantially higher inlet air pressure is used channeling may beencountered with resultant failure to develop the full strength of theinsecticide.

The specific examples herein should not be construed as unduly limitingthe invention.

I claim:

1. A process for producing a microbial insecticide which comprisesabsorbing on a particulate carrier substrate a nutrient inoculumcontaining Bacillus thu'ringien sis, the moisture content of saidsubstrate being about 55-62% by weight of the mixture, passing firststage air at a pressure not in excess of about five pounds per squareinch through said substrate-inoculated particles at a temperature offrom about 25 C. to about 35 C., said air having a relative humidity ofat least about 70%, for a period of time from about 25 hours to about 48hours until substantial sporulation occurs, thereafter passing secondstage air through the mass at a temperature substantially higher thanthat of said first stage air but not exceeding about 60 C. until themoisture content of the mass is reduced to a point below about 10% byweight,

said second stage air having a relative humidity substantially lowerthan that of said first stage air, and recovering the dried insecticide.

2. The process of claim 1 wherein the particulate material undergoingsporulation is maintained at a temperature of about 30 C. to about 34 C.

3. The process of claim 1 wherein the sporulation is carried out for aperiod of about 30 to about 48 hours.

4. The process of claim 1 wherein the first stage air has a relativehumidity of at least about 95%.

5. A process of claim 1 wherein the particulate substrate comprises 'amaterial of vegetable origin.

6. A process of claim 1 wherein the particulatesubstrate comprises afriable inorganic substance.

7. The process of claim 5 wherein the vegetable material is bran.

8. The process of claim 6 wherein the particulate substrate comprisesexpanded perlite.

9. A process of claim 1 wherein the first stage air is passed throughthe particulate mass at a rate of about 0.5 to about 1.0 volumes pervolume of said mass.

10. A process for producing a microbial insecticide which comprisesproviding a particulate carrier substance bearing on the surface thereofa nutrient medium for Bacillus thuringiensis, inoculating said medium onsaid carrier substance with Bacillus thuringiensis, passing first stageair at a pressure not in excess of about five p.s.i. into saidparticulate carrier substance bearing said inoculated medium untilsubstantial sporulation occurs, said first stage air being introducedinto said carrier substance at a temperature of from about 25 C. toabout 35 C. and at a relative humidity of at least about 70 maintainingparticulate carrier substance at a temperature of from about 25 C. toabout 35 C. substantially throughout the passage of said first stage airthereinto, thereafter passing second stage air having a relativehumidity substantially lower and a temperature not in excess of about 60C. but substantially higher than that of said first stage air throughsaid sporulated nutrient medium and carrier substance to reduce themoisture content thereof to not more than about 10% by weight.

11. The process of claim 10 wherein said first stage air is passed intosaid carrier substance at a temperature. of about 28 C. to about 30 C.

12. The process of claim 10 wherein the passage of said first stage airinto said carrier substance is discontinued after a time period of fromabout to about hours and wherein said process is completed in not morethan about hours.

13. The process of claim 10 wherein said first stage air is introducedinto carrier substance only after the temperature thereof has risen atleast about three to about five degrees centigrade after inoculation byreason of growth of Bacillus thuringiensis.

14. The process of claim 13 wherein said first stage air is introducedinto said inoculated nutrient medium-bearing carrier substance onlyafter the temperature thereof has reached at least about 35 C.

15. The process of claim 10 wherein the sporulated product, subsequentto the passage of second stage air therethrough, is comminuted toprovide a microbial insecticide.

16. The process of claim 10 wherein the passage of second stage airthrough said sporulated nutrient medium and carrier substance iscontinued until the moisture content thereof is reduced to not more than5% by weight.

17. The process of claim 1 in which the dried insecticide is furthercomminuted.

18. The process of claim 1 wherein the carrier substrate is a mixture ofparticulate expanded perlite and bran.

References Cited in the file of this patent Zinsser: Textbook ofBacteriology, 7th ed., 1935, pages 1 151-1152.

Chemical and Engineering News, 36: 51, December 22, 1958, page 15.

Fleschner: Science, vol. 129, number 3348, Feb. 27, 1959, pages 537-544;

Kushner et al.:- -J. Gen. Microbiology 21, 1959; page 96.

1. A PROCESS FOR PRODUCING A MICROBIAL INSECTICIDE WHICH COMPRISESABSORBING ON A PARTICULATE CARRIER SUBSTRATE A NUTRIENT INOCULUMCONTAING BACILLUS THURINGIENSIS, THE MOISTURE CONTENT OF SAID SUBSTRATEBEING ABOUT 55-62% BY WEIGHT OF THE MIXTURE, PASSING FIRST STAGE AIR ATA PRESSURE NOT IN EXCESS OF ABOUT FIVE POUNDS PER SQUARE INCH THROUGHSUCH SUBSTRATE-INOCULATED PARTICLES AT A TEMPERATURE OF FROM ABOUT 25*C.TO ABOUT 35*C., SAID AIR HAVING A RELATIVE HUMIDITY OF AT LEAST ABOUT70$ %, FOR A PERIOD OF TIME FROM ABOUT 25 HOURS TO ABOUT 48 HOURS UNTILSUBSTANTIAL SPORULATION OCCURS, THEREAFTER PASSING SECOND STAGE AIRTHROUGH THE MASS AT A TEMPERATURE SUBSTANTIALLY HIGHER THAN THAT OF SAIDFIRST STAGE AIR BUT NOT EXCEEDING ABOUT 60*C. UNTIL THE MOISTURE CONTENTOF THE MASS IS REDUCED TO A POINT BELOW ABOUT 10% BY WEIGHT SAID SECONDSTAGE AIR HABING A RELATIVE HUMIDITY SUBSTANTIALLY LOWER THAN THAT OFSAID FIRST STAGE AIR, AND RECOVERING THE DRIED INSECTICIDE.